Method of Controlling a Polypeptide Modification Reaction

ABSTRACT

The invention relates to a method of controlling a polypeptide modification reaction, in particular but not exclusively, a method of controlling the activation of human factor VII (FVII) to produce human factor VII(a) (FVII(a)). The invention also relates to polypeptides obtainable by the polypeptide modification reaction and to pharmaceutical compositions comprising said polypeptides.

FIELD OF THE INVENTION

The invention relates to a method of controlling a polypeptidemodification reaction, in particular but not exclusively, a method ofcontrolling the activation of human factor VII (FVII) to produce humanfactor VII(a) (FVII(a)). The invention also relates to polypeptidesobtainable by the polypeptide modification reaction and topharmaceutical compositions comprising said polypeptides.

BACKGROUND OF THE INVENTION

Blood coagulation is a process consisting of a complex interaction ofvarious blood components (or factors) that eventually gives rise to afibrin clot. Generally, the blood components, which participate in whathas been referred to as the coagulation “cascade”, are enzymaticallyinactive proteins (proenzymes or zymogens) that are converted toproteolytic enzymes by the action of an activator (which itself is anactivated clotting factor). Coagulation factors that have undergone sucha conversion are generally referred to as “active factors”, and aredesignated by the addition of the letter “a” to the name of thecoagulation factor (e.g. Factor VII(a)).

FVII (also known as Single chain FVII, unactivated FVII or zymogen) is asingle polypeptide chain, which upon proteolytic cleavage of the peptidebond between Arg152 and Ile153 is converted into the activated form:FVII(a). This reaction can be catalyzed by FVII(a) auto-proteolysis orby other enzymes such as FXa or Russel viper venom. Auto activation hasthe advantage that no enzyme needs to be added and physically removed atthe end of the process.

It is very important to be able to control the activation of FVIIcarefully, as the content of heavy chain degradation products (AA290 andAA315) increases dramatically once a proportion of activation of morethan 99% is reached (i.e. once the preferred substrate of Arg 152 hasbecome depleted). It is therefore crucial not to over-activate theproduct. A high proportion of activation e.g. above 94% is at the sametime desirable, leaving a rather narrow interval (e.g. 94-99%) whereboth a low degradation content and high activity can be obtained.

A certain amount of enzyme activation will take place concurrentlyduring purification of the enzyme. Furthermore, the levels of activationduring the purification process will inevitably vary due to variationsin starting material composition—mainly FVII(a) titer and hence columnload. Unexpected holding time during purification will also give rise tovariations in the levels of activation. During purification, the FVII(a)molecules will experience varying conditions with respect toconcentration, pH, temperature and residence time, which will result inpartial activation. Following purification, it has been observed thatthe proportion of activation has varied widely (e.g. 16% to 74%)depending upon the purification technique and conditions. This variationin the proportion of activation following purification creates asignificant problem with respect to conducting a standardised activationprocess following purification.

The proportion of activation of FVII can be calculated through knownprocedures, however, no real-time measurement technique currently existsand the known procedures typically have a duration of approximately 30minutes. Therefore, if the proportion of activation is approaching 99%upon sample removal for measurement, then this value will be exceeded bythe time the results are obtained. This will result in high levels ofthe undesirable heavy chain degradation products.

U.S. Pat. No. 4,286,056 (Baxter Travenol Lab) describes a method forproducing activated prothrombin complex concentrate which comprisescontrolling the degree of activation by determining the activation stateof the starting material and then varying at least one of the activationconditions in accordance with analyses of the progress of activation ofthe starting material to arrive at a predetermined activation level. WO2007/013993 (Maxygen Holdings Ltd) describes a method for activatingFVII to FVII(a) in solution, comprising addition of an amine compound,Ca²⁺, adjusting the final pH of the solution to about 7.2 to 8.6,incubating the resulting activation mixture at between about 2° C. andabout 25° C. for an amount of time sufficient to convert at least 90% ofthe scFVII to FVII(a). U.S. Pat. No. 4,456,591 (Baxter Travenol Lab)describes a process of administering to a patient having a clottingfactor defect such as a deficiency or inhibitor an effective hemostaticamount of a composition in which the sole effective, activatedhemostatic agent is factor VII(a).

There is thus a great need for providing an improved method fordetermining the optimum reaction time to provide desired levels ofmodified enzyme.

The current invention also provides for a purer protease product. Apurer product is less likely to result in anti-protease (antibody)formation in the patient.

Furthermore, tight control of the rate of protease activation ultimatelyresults in reduced waste in a production plant, as fewer productionbatches will be discarded when a greater number of batches meets thespecified requirements, in terms of purity and evenness of quality.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a methodof controlling a polypeptide modification reaction which comprises thesteps of calculating at least one process variable and applying saidvariable to a mathematical model in order to calculate the value of afurther process variable.

According to a second aspect of the invention there is provided a methodof performing a polypeptide modification reaction which comprises thesteps of:

-   -   (a) measuring the initial concentration of the polypeptide;    -   (b) measuring the initial proportion of modified polypeptide;    -   (c) calculating the polypeptide modification reaction time by        correlation of the values calculated in each of steps (a)        and (b) with a value of required proportion of modified        polypeptide; and    -   (d) performing the polypeptide modification reaction for the        time calculated in step (c).

According to a third aspect of the invention there is provided apolypeptide obtainable according to the method as defined hereinbefore.

DRAWINGS

FIG. 1 illustrates the overall concept of the invention. With variedinput, the process ensures that the output is essentially predictableand constant.

FIG. 2 illustrates the importance of closely regulating the degree towhich a protease is activated. As the degree of activation (%) increasesfrom 99.5% towards 100%, the percentage of degraded product (protease)increases exponentially.

FIG. 3 illustrates that the more enzyme (that is, protease) present in abatch, the faster activation of a protease will occur. The molarfraction (xb) stated as a percentage can be found using theHenderson-Hasselbach diagram. The active fraction of the enzyme can becalculated for any pH. There is nonlinear correlation.

FIG. 4 shows that the concentration of FVII(a) obtained is concentrationdependent. At high FVII(a) concentration, the rate of FVII activation isgreater than at low FVII(a) concentration.

FIG. 5 shows that the concentration of FVII(a) obtained is pH-dependent.At a higher pH (6.8) the rate of FVII activation is higher; at a lowerpH (6.2), the rate of FVII activation is lower and at pH 6.5 the rate ofFVII activation is intermediate.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention there is provided a methodof controlling a polypeptide modification reaction which comprises thesteps of calculating at least one process variable and applying saidvariable to a mathematical model in order to calculate the value of afurther process variable.

The invention therefore provides the benefit of combining a physical ormathematical model with a process analytical technology (PAT) tool inorder to evaluate an essential process variable. The PAT tool has theadvantage of being applied in an on-line, in-line and/or at-line mannerto accurately control the modification reaction. Thus, the use of such atechnique provides the user with an immediate required value such thatthe reaction can be performed in an optimum manner to achieve optimumresults. In one embodiment, the at least one process variable may beselected from degree of modification, reagent concentrationmeasurements, pH measurements, temperature measurements. In oneembodiment, the further process variable may be reaction time.

In one embodiment, the method comprises controlling the degree ofpegylation of a polypeptide. Thus according to a further aspect of theinvention there is provided a method of controlling the degree ofpegylation of a polypeptide which comprises the steps of applying thedegree of required pegylation, enzyme concentration, PEG concentration,polypeptide concentration and temperature to a mathematical model andcalculating the reaction time.

In one embodiment, the method comprises controlling the degree ofpegylation of a factor IX (FIX) polypeptide. In one embodiment, thedegree of pegylation is calculated in accordance with the followingprocess variables: reaction time, enzyme concentration, PEGconcentration, FIX concentration and temperature (which will typicallybe 22° C.). It will be appreciated that the skilled person will be ableto calculate the optimum reaction time to achieve a required degree ofpegylation of FIX by inputting values of the process variables into amathematical model (which may be derived from the Eulers, Runge-Kutta,Newton-Raphson or DASPK methods). Such mathematical methods can beadvantageously employed to provide accurate control of polypeptidemodification reactions.

According to a second aspect of the invention there is provided a methodof performing a polypeptide modification reaction which comprises thesteps of:

-   -   (a) measuring the initial concentration of the polypeptide;    -   (b) measuring the initial proportion of modified polypeptide;    -   (c) calculating the polypeptide modification reaction time by        correlation of the values calculated in each of steps (a)        and (b) with a value of required proportion of modified        polypeptide; and    -   (d) performing the polypeptide modification reaction for the        time calculated in step (c).

In one embodiment, the modification reaction comprises enzymaticcleavage or modification by the addition of a chemical agent to apolypeptide (e.g. pegylation).

In the embodiment wherein modification comprises enzymatic cleavage, ithas been surprisingly found that the initial concentration andproportion of cleavage can be correlated with the required proportion ofcleavage in order to calculate a precise reaction time. The results ofthis correlation are extremely accurate (often to within approximately0.5% proportion of cleavage) and repeatable. The process also providesthe significant advantage that only two measurements need to becalculated prior to reaction (namely initial concentration andproportion of cleavage). Furthermore, the reaction may proceed for thecalculated time without the need for monitoring the state of thereaction (or even measuring the final proportion of cleavage, unlessrequired for quality control purposes).

The term “protein”, “polypeptide” and “peptide” as used herein means acompound composed of at least five constituent amino acids connected bypeptide bonds. The constituent amino acids may be from the group of theamino acids encoded by the genetic code and they may be natural aminoacids which are not encoded by the genetic code, as well as syntheticamino acids. Natural amino acids which are not encoded by the geneticcode are e.g. hydroxyproline, y-carboxyglutamate, ornithine,phosphoserine, D-alanine and D-glutamine. Synthetic amino acids compriseamino acids manufactured by chemical synthesis, i.e. D-isomers of theamino acids encoded by the genetic code such as D-alanine and D-leucine,Aib (a-aminoisobutyric acid), Abu (a-aminobutyric acid), Tle(tert-butylglycine), β-alanine, 3-aminomethyl benzoic acid andanthranilic acid.

In one embodiment, the polypeptide is an enzyme, such as a bloodcoagulation factor or hemostasis related related protein. (e.g. a serineprotease). Examples of such polypeptides include: I (fibrinogen), II(prothrombin), tissue factor, V (proaccelerin), VI, VII, VIII, IX(Christmas factor), X (Stuart-Prower factor), XI (plasma thromboplastinantecedent), XII (Hageman factor), XIII (fibrin-stabilizing factor), vonWillebrand factor, prekallikrein, high molecular weight kininogen(HMWK), fibronectin, antithrombin III, heparin cofactor II, protein C,protein S, protein Z, protein Z-related protease inhibitor (ZPI),plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA),urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogenactivator inhibitor-2 (PAI2) and cancer procoagulant.

In a further embodiment, the polypeptide is an autoactivatedpolypeptide. In a further embodiment, the enzyme is a blood coagulationfactor (e.g. a serine protease blood coagulation factor). Examples ofsuitable serine protease blood coagulation factors include thoseclassified under EC 3.4.21, for example: II, VII, IX, X, XI, XII,prekallikrein, protein C and plasminogen (the activated forms of theseinactive zymogens are FIIa, FVIIa, FIXa, FXa, FXIa, FXIIa, kallikrein,activated protein C (aPC) and plasmin, respectively).

In one embodiment wherein the modification reaction comprises enzymaticcleavage, the blood coagulation factor is factor VII or an analogue orderivative thereof.

In one embodiment wherein the modification reaction comprisespegylation, the blood coagulation factor is factor IX or an analogue orderivative thereof.

In one aspect of the invention, the invention provides a method ofactivating a serine protease blood coagulation factor which comprisesthe steps of:

-   -   (a) measuring the initial concentration of the serine protease        blood coagulation factor;    -   (b) measuring the initial proportion of activated serine        protease blood coagulation factor;    -   (c) calculating the serine protease blood coagulation factor        activation reaction time by correlation of the values measured        in each of steps (a) and (b) with a value of required proportion        of activated serine protease blood coagulation factor; and    -   (d) performing the serine protease blood coagulation factor        activation reaction for the time calculated in step (c);    -   (e) terminating the reaction after the reaction time calculated        in step (c).

According to a further aspect, the invention provides a method ofactivating factor VII to factor VII(a), or an analogue or derivativethereof, which comprises the steps of:

-   -   (a) measuring the initial concentration of factor VII;    -   (b) measuring the initial proportion of activated factor VII;    -   (c) calculating the factor VII activation reaction time by        correlation of the values calculated in each of steps (a)        and (b) with a value of required proportion of activated factor        VII; and    -   (d) performing the factor VII activation reaction for the time        calculated in step (c).

In a further optional step (e), the reaction is terminated after thereaction time calculated in step (c).

In a still further aspect, the invention provides a method of preventingdegradation of an activated serine protease product which comprises thesteps of:

-   -   (a) measuring the initial concentration of the serine protease        blood coagulation factor;    -   (b) measuring the initial proportion of activated serine        protease blood coagulation factor;    -   (c) calculating the serine protease blood coagulation factor        activation reaction time by correlation of the values measured        in each of steps (a) and (b) with a value of required proportion        of activated serine protease blood coagulation factor; and    -   (d) performing the serine protease blood coagulation factor        activation reaction for the time calculated in step (c);    -   (e) terminating the reaction after the reaction time calculated        in step (c).

In one embodiment of the invention, the correlation procedure describedin step (c) is calculated in accordance with formula (I):

$\begin{matrix}{t = \frac{- {\ln \left( \frac{{akt}\; {0 \cdot \left( {{akt} - 1} \right)}}{{akt} \cdot \left( {{{akt}\; 0} - 1} \right)} \right)}}{{{k(T)} \cdot {xb} \cdot F}\; 0}} & (I)\end{matrix}$

wherein “akt” refers to the required proportion of cleaved polypeptide,“akt0” refers to the initial proportion of cleaved polypeptide measuredin step (b), “F0” refers to the initial concentration of the polypeptide(in g/l) measured in step (a), k(T) refers to the reaction constant forthe given reaction (in L/g/min) as a function of temperature, T, and xbrefers to the molar fraction. In one embodiment, k(T) is a polynomial ora spline which describes the variation of k with temperature. ForrFVIIa, the following 3rd order polynomial was used: k(T)=k*(0.00001T̂3−0.00147 T̂2+0.02566 T+0.86729) with T being the temperature (5-60°C.). In a similar fashion, pKa can be expressed as a function oftemperature.

In one embodiment, the temperature is between 5° C. and 25° C.,preferably 10° C. to 20° C. In a further embodiment, the activationreaction is performed at room temperature (e.g. approximately 21.5° C.).

In the embodiment wherein the modification comprises cleavage of factorVII, the activation reaction is typically performed at a constanttemperature.

Application of the invention to the activation of factor VIIbeneficially results in the production of fully activated factor VIImolecules containing a minimum of degradation products.

The term “analogue” as used herein referring to a polypeptide means amodified peptide wherein one or more amino acid residues of the peptidehave been substituted by other amino acid residues and/or wherein one ormore amino acid residues have been deleted from the peptide and orwherein one or more amino acid residues have been added to the peptide.Such addition or deletion of amino acid residues can take place at theN-terminal of the peptide and/or at the C-terminal of the peptide. Allamino acids for which the optical isomer is not stated are to beunderstood to mean the L-isomer.

Examples of factor VII analogues may be found in WO 02/22776, theanalogues of which are herein incorporated by reference. In oneembodiment, the factor VII analogue is a hyperactive analogue, i.e. onewhich has at least two fold greater amidolytic activity than wild-typefactor VII. In a preferred embodiment, the factor VII analogue isV158D/E296V/M298Q-FVII(a) (Example 6 in WO 02/22776).

Measurement of the initial concentration of the polypeptide in step (a)may typically be performed by UV spectroscopy. In one embodiment, theconcentration will be adjusted to between approximately 1.5 g/L and 2.2g/L (e.g approximately 1.9 g/L).

Measurement of the initial proportion of cleaved polypeptide in step (b)may typically be performed by reduced SDS-PAGE, reduced or non-reducedHPLC or chip electrophoresis (e.g. Agilent Bioanalyzer). In oneembodiment, step (b) is performed by chip electrophoresis (e.g. AgilentBioanalyzer). In the embodiment wherein the polypeptide comprises factorVII, the initial proportion of activated factor VII(a) will typically bebetween 10 and 90% depending upon the purification conditions.

It will be appreciated that references to “required proportion ofmodified polypeptide” refer to any proportion of modification requiredby the user. In the embodiment wherein the modification comprisescleavage of factor VII, the required proportion of cleavage will bebetween 94 and 99%, such as between 95 and 97% (e.g. approximately 95%).These ranges would typically be selected to provide a high proportion ofactivation products (e.g. factor VII(a)) but minimise the amount ofheavy chain degradation products (AA290 and AA315).

In the embodiment wherein the modification comprises cleavage of factorVII, the cleavage reaction additionally comprises the addition of anamine compound (e.g.

histidine, Tris, lysine, arginine, phosphorylcholine, or betaine). In afurther embodiment, the amine compound is histidine. In one embodiment,the amine compound is added to a final concentration of about 1 to 500mM, such as about 10 to 100 mM (e.g. 10 mM).

In the embodiment wherein the modification comprises cleavage of factorVII, the cleavage reaction additionally comprises the addition ofcalcium ions (e.g. calcium chloride). In one embodiment, the calciumions are added to a final concentration of about 1 to 50 mM, such asbetween 10 and 25 mM (e.g. 12 mM).

In the embodiment wherein the modification comprises cleavage of factorVII, the cleavage reaction additionally comprises the addition of sodiumchloride. In one embodiment, the sodium chloride is added to a finalconcentration of about 1 to 100 mM, such as between 20 and 80 mM (e.g.60 mM).

In one embodiment of the invention, the required proportion of serineprotease cleavage is between 90 and 99%, such as between 94 and 99%,such as between 95 and 97%, such as between 96 and 98%, such as between97 and 99%.

In the embodiment wherein the modification comprises cleavage of factorVII, the activation reaction is typically performed at a pH of between6.0 and 8.0. The autocatalytic reaction of factor VII is pH dependent.As the pH is raised, the amidolytic activity of factor VII is initiatedand therefore the reaction rate will increase accordingly. It istherefore desirable to choose a pH value between 6.0 and 8.0 and ensurethat the reaction proceeds at this pH by appropriate buffering. If thepH varies during the activation reaction then the rate of activationwill vary from that calculated in step (c) and therefore impact upon thequality of the resultant product.

Thus, in one embodiment, the method of the invention additionallycomprises the step of selecting a pH of between 6.0 and 8.0 prior toinitiation of the activation reaction and maintaining the reaction atthe selected pH value during the activation reaction. In a furtherembodiment, the pH is selected from between 6.25 and 6.75 (e.g.6.5±0.05).

In view of the substantial accuracy of the methodology described herein,it is desirable to ensure that the activation reaction is terminatedimmediately after the reaction time calculated in step (c). If theactivation reaction is allowed to continue beyond the time calculated instep (c) then this will increase the potential presence of undesirableheavy chain degradation products. A number of alternative methods fortermination are known to those skilled in the art, for example, theaddition of silica to the reaction mixture. However, in one embodiment,the activation reaction is terminated by lowering the pH to a valuebelow about 6.0, such as between 5.5 and 6.0 (e.g. 5.8). In oneembodiment, the pH is lowered by the addition of a strong acid (e.g. 1Mhydrochloric acid).

In a further embodiment of the invention, the correlation proceduredescribed in step (c), used to calculate the reaction time (hereinafterreferred to as “t”) may be calculated in accordance with formula (II):

$\begin{matrix}{t = \frac{- {\ln \left( \frac{{akt}\; {0 \cdot \left( {{akt} - 1} \right)}}{{akt} \cdot \left( {{{akt}\; 0} - 1} \right)} \right)}}{{k \cdot {xb} \cdot F}\; 0}} & ({II})\end{matrix}$

wherein “akt” refers to the required proportion of cleaved polypeptide,“akt0” refers to the initial proportion of cleaved polypeptide measuredin step (b), “F0” refers to the initial concentration of the polypeptide(in g/l) measured in step (a), k refers to the reaction constant for thegiven reaction (in L/g/min) and xb refers to the molar fraction.

The molar fraction (xb) can be calculated using the Henderson-Hasselbachrelationship which correlates between the active fraction of the enzymeat any pH value. Typically, this relationship will be a non-linearcorrelation (e.g. sigmoidal).

In the embodiment wherein the modification comprises cleavage of factorVII, xb may be calculated based on the degree of protonisation ofhistidine. Serine proteases (including factor VII(a)) are characterisedby a catalytic triad consisting of three residues: Serine 139, Histidine57 and Aspartate 81. It is known that the histidine residue must bedeprotonised in order to react and the serine protease is only active ina pH range above the pKa of histidine (which is 7.61).

Therefore, in the embodiment wherein the modification comprises cleavageof factor VII, xb may be calculated according to the following equationin formula (III):

$\begin{matrix}{{xb} = \frac{10^{{pH} - 7.61}}{1 + 10^{{pH} - 7.61}}} & ({III})\end{matrix}$

wherein pH refers to the selected pH of the reaction.

The value k may be calculated in accordance with the reaction kineticsof the given reaction intended to be measured. Thus, k is a physicalconstant which defines concentration dependency. Such a constant maygenerally be calculated in accordance with the sum of least squareswhich will be readily apparent to those skilled in the art.

For example, in the embodiment wherein the polypeptide comprises factorVII, the value of k has been calculated as 0.29 L/g/min. Therefore, inthe embodiment wherein the modification comprises cleavage of factorVII, the correlation procedure described in step (c) to calculate thereaction time (“t”) may be calculated in accordance with formula (IV):

$\begin{matrix}{t = \frac{- {\ln \left( \frac{{akt}\; {0 \cdot \left( {{akt} - 1} \right)}}{{akt} \cdot \left( {{{akt}\; 0} - 1} \right)} \right)}}{{0.29 \cdot {xb} \cdot F}\; 0}} & ({IV})\end{matrix}$

Wherein akt, akt0, xb and F0 are as hereinbefore defined.

In a further embodiment of the invention, the correlation proceduredescribed in step (c) is calculated by means of formula (V), in which xbof formula (II) is 1:

$\begin{matrix}{t = \frac{- {\ln \left( \frac{{akt}\; {0 \cdot \left( {{akt} - 1} \right)}}{{akt} \cdot \left( {{{akt}\; 0} - 1} \right)} \right)}}{{k \cdot F}\; 0}} & (V)\end{matrix}$

wherein “akt” refers to the required proportion of cleaved polypeptide,“akt0” refers to the initial proportion of cleaved polypeptide measuredin step (b), “F0” refers to the initial concentration of the polypeptide(in g/l) measured in step (a) and k refers to the reaction constant forthe given reaction (in L/g/min).

According to a third aspect of the invention there is provided apolypeptide obtainable according to the method as defined hereinbefore.

In different embodiments, said blood coagulation serine protease isFactor II or Factor VII or Factor IX or Factor X or Factor XI or FactorXII; or an analogue or derivative of any one of said blood coagulationfactors.

In one embodiment, the polypeptide is a factor VII(a) or factor IXanalogue or derivative.

The factor VII(a) or factor IX analogues or derivatives andpharmaceutical compositions comprising the factor VII(a) or factor IXanalogues or derivatives according to the present invention may be usedin the treatment of diseases alleviated by administration of humancoagulation factor VII(a) or IX, such as a bleeding disorder e.g.hemophilia, a blood disease, hemarthrosis, hematomas, mucocutaneousbleeding, inherited blood disease, familial bleeding disorder, familialblood disease or factor replacement therapy. In one embodiment, thedisease alleviated by administration of human coagulation factor VII(a)or IX is hemophilia, such as hemophilia B or Christmas disease.

Thus according to a further aspect of the invention there is provided amethod of treating hemophilia which comprises administering to a patienta therapeutically effective amount of a factor VII(a) or IX analogue orderivative as defined hereinbefore.

There is also provided a factor VII(a) or a factor IX analogue orderivative, as defined hereinbefore, for use in the treatment ofhemophilia.

There is also provided the use of a factor VII(a) or a factor IXanalogue or derivative as defined hereinbefore in the manufacture of amedicament for the treatment of hemophilia.

There is also provided a pharmaceutical composition comprising a factorVII(a) or IX analogue or derivative as defined hereinbefore for use inthe treatment of hemophilia.

The term “treatment” and “treating” as used herein means the managementand care of a patient for the purpose of combating a condition, such asa disease or a disorder. The term is intended to include the fullspectrum of treatments for a given condition from which the patient issuffering, such as administration of the active compound to alleviatethe symptoms or complications, to delay the progression of the disease,disorder or condition, to alleviate or relief the symptoms andcomplications, and/or to cure or eliminate the disease, disorder orcondition as well as to prevent the condition, wherein prevention is tobe understood as the management and care of a patient for the purpose ofcombating the disease, condition, or disorder and includes theadministration of the active peptides to prevent the onset of thesymptoms or complications. The patient to be treated is preferably amammal, in particular a human being, but it may also include animals,such as dogs, cats, cows, sheep and pigs. It is to be understood, thattherapeutic and prophylactic (preventive) regimes represent separateaspects of the present invention.

A “therapeutically effective amount” of a peptide as used herein meansan amount sufficient to cure, alleviate or partially arrest the clinicalmanifestations of a given disease and its complications. An amountadequate to accomplish this is defined as “therapeutically effectiveamount”. Effective amounts for each purpose will depend on the type andseverity of the disease or injury as well as the weight and generalstate of the subject. It will be understood that determining anappropriate dosage may be achieved using routine experimentation, byconstructing a matrix of values and testing different points in thematrix, which is all within the ordinary skills of a trained physicianor veterinary.

According to a further aspect of the invention, there is provided apharmaceutical formulation comprising a polypeptide as hereinbeforedefined.

The formulation may further comprise a buffer system, preservative(s),tonicity agent(s), chelating agent(s), stabilizers and surfactants. Inone embodiment of the invention the pharmaceutical formulation is anaqueous formulation, i.e. formulation comprising water. Such formulationis typically a solution or a suspension. In one embodiment of theinvention the pharmaceutical formulation is an aqueous solution.

The term “aqueous formulation” is defined as a formulation comprising atleast 50% w/w water. Likewise, the term “aqueous solution” is defined asa solution comprising at least 50% w/w water, and the term “aqueoussuspension” is defined as a suspension comprising at least 50% w/wwater.

In one embodiment the pharmaceutical formulation is a freeze-driedformulation, whereto the physician or the patient adds solvents and/ordiluents prior to use.

In one embodiment the pharmaceutical formulation is a dried formulation(e.g. freeze-dried or spray-dried) ready for use without any priordissolution.

In one embodiment the invention relates to a pharmaceutical formulationcomprising an aqueous solution of a peptide of the present invention,and a buffer, wherein said peptide is present in a concentration from0.1-100 mg/ml, and wherein said formulation has a pH from about 2.0 toabout 10.0.

In one embodiment of the invention the pH of the formulation is selectedfrom the list consisting of 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8,2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2,4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6,5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4,8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8,9.9, and 10.0.

In one embodiment of the invention the buffer is selected from the groupconsisting of sodium acetate, sodium carbonate, citrate, glycylglycine,histidine, glycine, lysine, arginine, sodium dihydrogen phosphate,disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention.

In one embodiment of the invention the formulation further comprises apharmaceutically acceptable preservative. In one embodiment of theinvention the preservative is selected from the group consisting ofphenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propylp-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate,2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal,bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate,chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride,chlorphenesine (3p-chlorphenoxypropane-1,2-diol) or mixtures thereof.

In one embodiment of the invention the preservative is present in aconcentration from 0.1 mg/ml to 20 mg/ml. In one embodiment of theinvention the preservative is present in a concentration from 0.1 mg/mlto 5 mg/ml. In one embodiment of the invention the preservative ispresent in a concentration from 5 mg/ml to 10 mg/ml. In one embodimentof the invention the preservative is present in a concentration from 10mg/ml to 20 mg/ml. Each one of these specific preservatives constitutesan alternative embodiment of the invention. The use of a preservative inpharmaceutical compositions is well-known to the skilled person. Forconvenience reference is made to Remington: The Science and Practice ofPharmacy, 20^(th) edition, 2000.

In one embodiment of the invention the formulation further comprises anisotonic agent. In one embodiment of the invention the isotonic agent isselected from the group consisting of a salt (e.g. sodium chloride), asugar or sugar alcohol, an amino acid (e.g. L-glycine, L-histidine,arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), analditol (e.g. glycerol (glycerine), 1,2-propanediol (propyleneglycol),1,3-propanediol, 1,3-butanediol) polyethyleneglycol (e.g. PEG400), ormixtures thereof. Any sugar such as mono-, di-, or polysaccharides, orwater-soluble glucans, including for example fructose, glucose, mannose,sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran,pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch andcarboxymethylcellulose-Na may be used. In one embodiment the sugaradditive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbonhaving at least one —OH group and includes, for example, mannitol,sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In oneembodiment the sugar alcohol additive is mannitol. The sugars or sugaralcohols mentioned above may be used individually or in combination.There is no fixed limit to the amount used, as long as the sugar orsugar alcohol is soluble in the liquid preparation and does notadversely effect the stabilizing effects achieved using the methods ofthe invention. In one embodiment, the sugar or sugar alcoholconcentration is between about 1 mg/ml and about 150 mg/ml. In oneembodiment of the invention the isotonic agent is present in aconcentration from 1 mg/ml to 50 mg/ml. In one embodiment of theinvention the isotonic agent is present in a concentration from 1 mg/mlto 7 mg/ml. In one embodiment of the invention the isotonic agent ispresent in a concentration from 8 mg/ml to 24 mg/ml. In one embodimentof the invention the isotonic agent is present in a concentration from25 mg/ml to 50 mg/ml. Each one of these specific isotonic agentsconstitutes an alternative embodiment of the invention. The use of anisotonic agent in pharmaceutical compositions is well-known to theskilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 20^(th) edition, 2000.

In one embodiment of the invention the formulation further comprises achelating agent. In one embodiment of the invention the chelating agentis selected from salts of ethylenediaminetetraacetic acid (EDTA), citricacid, and aspartic acid, and mixtures thereof. In one embodiment of theinvention the chelating agent is present in a concentration from 0.1mg/ml to 5 mg/ml. In one embodiment of the invention the chelating agentis present in a concentration from 0.1 mg/ml to 2 mg/ml. In oneembodiment of the invention the chelating agent is present in aconcentration from 2 mg/ml to 5 mg/ml. Each one of these specificchelating agents constitutes an alternative embodiment of the invention.The use of a chelating agent in pharmaceutical compositions iswell-known to the skilled person. For convenience reference is made toRemington: The Science and Practice of Pharmacy, 20^(th) edition, 2000.

In one embodiment of the invention the formulation further comprises astabilizer. The use of a stabilizer in pharmaceutical compositions iswell-known to the skilled person. For convenience reference is made toRemington: The Science and Practice of Pharmacy, 20^(th) edition, 2000.

More particularly, compositions of the invention are stabilized liquidpharmaceutical compositions whose therapeutically active componentsinclude a polypeptide that possibly exhibits aggregate formation duringstorage in liquid pharmaceutical formulations. By “aggregate formation”is intended a physical interaction between the polypeptide moleculesthat results in formation of oligomers, which may remain soluble, orlarge visible aggregates that precipitate from the solution. By “duringstorage” is intended a liquid pharmaceutical composition or formulationonce prepared, is not immediately administered to a subject. Rather,following preparation, it is packaged for storage, either in a liquidform, in a frozen state, or in a dried form for later reconstitutioninto a liquid form or other form suitable for administration to asubject. By “dried form” is intended the liquid pharmaceuticalcomposition or formulation is dried either by freeze drying (i.e.,lyophilization; see, for example, Williams and Polli (1984) J.Parenteral Sci. Technol. 38:48-59), spray drying (see Masters (1991) inSpray-Drying Handbook (5th ed; Longman Scientific and Technical, Essez,U.K.), pp. 491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm.18:1169-1206; and Mumenthaler et al. (1994) Pharm. Res. 11:12-20), orair drying (Carpenter and Crowe (1988) Cryobiology 25:459-470; and Roser(1991) Biopharm. 4:47-53). Aggregate formation by a polypeptide duringstorage of a liquid pharmaceutical composition can adversely affectbiological activity of that polypeptide, resulting in loss oftherapeutic efficacy of the pharmaceutical composition. Furthermore,aggregate formation may cause other problems such as blockage of tubing,membranes, or pumps when the polypeptide-containing pharmaceuticalcomposition is administered using an infusion system.

The pharmaceutical compositions of the invention may further comprise anamount of an amino acid base sufficient to decrease aggregate formationby the polypeptide during storage of the composition. By “amino acidbase” is intended an amino acid or a combination of amino acids, whereany given amino acid is present either in its free base form or in itssalt form. Where a combination of amino acids is used, all of the aminoacids may be present in their free base forms, all may be present intheir salt forms, or some may be present in their free base forms whileothers are present in their salt forms. In one embodiment, amino acidsto use in preparing the compositions of the invention are those carryinga charged side chain, such as arginine, lysine, aspartic acid, andglutamic acid. Any stereoisomer (i.e., L, D, or mixtures thereof) of aparticular amino acid (e.g. glycine, methionine, histidine, imidazole,arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine andmixtures thereof) or combinations of these stereoisomers, may be presentin the pharmaceutical compositions of the invention so long as theparticular amino acid is present either in its free base form or itssalt form. In one embodiment the L-stereoisomer is used. Compositions ofthe invention may also be formulated with analogues of these aminoacids. By “amino acid analogue” is intended a derivative of thenaturally occurring amino acid that brings about the desired effect ofdecreasing aggregate formation by the polypeptide during storage of theliquid pharmaceutical compositions of the invention. Suitable arginineanalogues include, for example, aminoguanidine, ornithine andN-monoethyl L-arginine, suitable methionine analogues include ethionineand buthionine and suitable cysteine analogues include S-methyl-Lcysteine. As with the other amino acids, the amino acid analogues areincorporated into the compositions in either their free base form ortheir salt form. In one embodiment of the invention the amino acids oramino acid analogues are used in a concentration, which is sufficient toprevent or delay aggregation of the protein.

In one embodiment of the invention methionine (or other sulphuric aminoacids or amino acid analogous) may be added to inhibit oxidation ofmethionine residues to methionine sulfoxide when the polypeptide actingas the therapeutic agent is a polypeptide comprising at least onemethionine residue susceptible to such oxidation. By “inhibit” isintended minimal accumulation of methionine oxidized species over time.Inhibiting methionine oxidation results in greater retention of thepolypeptide in its proper molecular form. Any stereoisomer of methionine(L, D, or mixtures thereof) or combinations thereof can be used. Theamount to be added should be an amount sufficient to inhibit oxidationof the methionine residues such that the amount of methionine sulfoxideis acceptable to regulatory agencies. Typically, this means that thecomposition contains no more than about 10% to about 30% methioninesulfoxide. Generally, this can be achieved by adding methionine suchthat the ratio of methionine added to methionine residues ranges fromabout 1:1 to about 1000:1, such as 10:1 to about 100:1.

In one embodiment of the invention the formulation further comprises astabilizer selected from the group of high molecular weight polymers orlow molecular compounds. In one embodiment of the invention thestabilizer is selected from polyethylene glycol (e.g. PEG 3350),polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy-/hydroxycelluloseor derivates thereof (e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins,sulphur-containing substances as monothioglycerol, thioglycolic acid and2-methylthioethanol, and different salts (e.g. sodium chloride). Eachone of these specific stabilizers constitutes an alternative embodimentof the invention.

The pharmaceutical compositions may also comprise additional stabilizingagents, which further enhance stability of a therapeutically activepolypeptide therein. Stabilizing agents of particular interest to thepresent invention include, but are not limited to, methionine and EDTA,which protect the polypeptide against methionine oxidation, and anonionic surfactant, which protects the polypeptide against aggregationassociated with freeze-thawing or mechanical shearing.

In one embodiment of the invention the formulation further comprises asurfactant. In one embodiment of the invention the surfactant isselected from a detergent, ethoxylated castor oil, polyglycolyzedglycerides, acetylated monoglycerides, sorbitan fatty acid esters,polyoxypropylene-polyoxyethylene block polymers (eg. poloxamers such asPluronic® F68, poloxamer 188 and 407, Triton X-100), polyoxyethylenesorbitan fatty acid esters, polyoxyethylene and polyethylene derivativessuch as alkylated and alkoxylated derivatives (tweens, e.g. Tween-20,Tween-40, Tween-80 and Brij-35), monoglycerides or ethoxylatedderivatives thereof, diglycerides or polyoxyethylene derivativesthereof, alcohols, glycerol, lectins and phospholipids (eg. phosphatidylserine, phosphatidyl choline, phosphatidyl ethanolamine, phosphatidylinositol, diphosphatidyl glycerol and sphingomyelin), derivates ofphospholipids (eg. dipalmitoyl phosphatidic acid) and lysophospholipids(eg. palmitoyl lysophosphatidyl-L-serine and1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine) and alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, and the positively charged DODAC, DOTMA,DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, andglycerophospholipids (eg. cephalins), glyceroglycolipids (eg.galactopyransoide), sphingoglycolipids (eg. ceramides, gangliosides),dodecylphosphocholine, hen egg lysolecithin, fusidic acid derivatives-(e.g. sodium tauro-dihydrofusidate etc.), long-chain fatty acids andsalts thereof C6-C12 (eg. oleic acid and caprylic acid), acylcarnitinesand derivatives, N^(α)-acylated derivatives of lysine, arginine orhistidine, or side-chain acylated derivatives of lysine or arginine,N^(α)-acylated derivatives of dipeptides comprising any combination oflysine, arginine or histidine and a neutral or acidic amino acid,N^(α)-acylated derivative of a tripeptide comprising any combination ofa neutral amino acid and two charged amino acids, DSS (docusate sodium,CAS registry no [577-11-7]), docusate calcium, CAS registry no[128-49-4]), docusate potassium, CAS registry no [7491-09-0]), SDS(sodium dodecyl sulphate or sodium lauryl sulphate), sodium caprylate,cholic acid or derivatives thereof, bile acids and salts thereof andglycine or taurine conjugates, ursodeoxycholic acid, sodium cholate,sodium deoxycholate, sodium taurocholate, sodium glycocholate,N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic(alkyl-aryl-sulphonates) monovalent surfactants, zwitterionicsurfactants (e.g. N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate, cationicsurfactants (quaternary ammonium bases) (e.g. cetyl-trimethylammoniumbromide, cetylpyridinium chloride), non-ionic surfactants (eg. Dodecylβ-D-glucopyranoside), poloxamines (eg. Tetronic's), which aretetrafunctional block copolymers derived from sequential addition ofpropylene oxide and ethylene oxide to ethylenediamine, or the surfactantmay be selected from the group of imidazoline derivatives, or mixturesthereof. Each one of these specific surfactants constitutes analternative embodiment of the invention.

The use of a surfactant in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 20^(th) edition, 2000.

It is possible that other ingredients may be present in the peptidepharmaceutical formulation of the present invention. Such additionalingredients may include wetting agents, emulsifiers, antioxidants,bulking agents, tonicity modifiers, chelating agents, metal ions,oleaginous vehicles, proteins (e.g., human serum albumin, gelatine orproteins) and a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine). Such additionalingredients, of course, should not adversely affect the overallstability of the pharmaceutical formulation of the present invention.

Pharmaceutical compositions containing a peptide of the presentinvention may be administered to a patient in need of such treatment atseveral sites, for example, at topical sites, for example, skin andmucosal sites, at sites which bypass absorption, for example,administration in an artery, in a vein, in the heart, and at sites whichinvolve absorption, for example, administration in the skin, under theskin, in a muscle or in the abdomen.

Administration of pharmaceutical compositions according to the inventionmay be through several routes of administration, for example, lingual,sublingual, buccal, in the mouth, oral, in the stomach and intestine,nasal, pulmonary, for example, through the bronchioles and alveoli or acombination thereof, epidermal, dermal, transdermal, vaginal, rectal,ocular, for examples through the conjunctiva, uretal, and parenteral topatients in need of such a treatment.

Compositions of the current invention may be administered in severaldosage forms, for example, as solutions, suspensions, emulsions,microemulsions, multiple emulsion, foams, salves, pastes, plasters,ointments, tablets, coated tablets, rinses, capsules, for example, hardgelatine capsules and soft gelatine capsules, suppositories, rectalcapsules, drops, gels, sprays, powder, aerosols, inhalants, eye drops,ophthalmic ointments, ophthalmic rinses, vaginal pessaries, vaginalrings, vaginal ointments, injection solution, in situ transformingsolutions, for example in situ gelling, in situ setting, in situprecipitating, in situ crystallization, infusion solution, and implants.

Compositions of the invention may further be compounded in, or attachedto, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug delivery system and advanced drugdelivery system in order to further enhance stability of the peptide ofthe present invention, increase bioavailability, increase solubility,decrease adverse effects, achieve chronotherapy well known to thoseskilled in the art, and increase patient compliance or any combinationthereof. Examples of carriers, drug delivery systems and advanced drugdelivery systems include, but are not limited to, polymers, for examplecellulose and derivatives, polysaccharides, for example dextran andderivatives, starch and derivatives, poly(vinyl alcohol), acrylate andmethacrylate polymers, polylactic and polyglycolic acid and blockco-polymers thereof, polyethylene glycols, carrier proteins, for examplealbumin, gels, for example, thermogelling systems, for example blockco-polymeric systems well known to those skilled in the art, micelles,liposomes, microspheres, nanoparticulates, liquid crystals anddispersions thereof, L2 phase and dispersions there of, well known tothose skilled in the art of phase behaviour in lipid-water systems,polymeric micelles, multiple emulsions, self-emulsifying,self-microemulsifying, cyclodextrins and derivatives thereof, anddendrimers.

Compositions of the current invention are useful in the formulation ofsolids, semisolids, powder and solutions for pulmonary administration ofa peptide of the present invention, using, for example a metered doseinhaler, dry powder inhaler and a nebulizer, all being devices wellknown to those skilled in the art.

Compositions of the current invention are specifically useful in theformulation of controlled, sustained, protracting, retarded, and slowrelease drug delivery systems. More specifically, but not limited to,compositions are useful in formulation of parenteral controlled releaseand sustained release systems (both systems leading to a many-foldreduction in number of administrations), well known to those skilled inthe art. Even more preferably, are controlled release and sustainedrelease systems administered subcutaneous. Without limiting the scope ofthe invention, examples of useful controlled release system andcompositions are hydrogels, oleaginous gels, liquid crystals, polymericmicelles, microspheres and nanoparticles.

Methods to produce controlled release systems useful for compositions ofthe current invention include, but are not limited to, crystallization,condensation, co-crystallization, precipitation, co-precipitation,emulsification, dispersion, high pressure homogenisation, encapsulation,spray drying, microencapsulating, coacervation, phase separation,solvent evaporation to produce microspheres, extrusion and supercriticalfluid processes. General reference is made to Handbook of PharmaceuticalControlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) andDrug and the Pharmaceutical Sciences vol. 99: Protein Formulation andDelivery (MacNally, E. J., ed. Marcel Dekker, New York, 2000).

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump. A furtheroption is a composition which may be a solution or suspension for theadministration of the peptide of the present inventionin the form of anasal or pulmonal spray. As a still further option, the pharmaceuticalcompositions containing the peptide of the present invention can also beadapted to transdermal administration, e.g. by needle-free injection orfrom a patch, optionally an iontophoretic patch, or transmucosal, e.g.buccal, administration.

The invention will now be described with reference to the followingnon-limited Examples:

Methodology Calculation of Initial Proportion of Activation

The initial proportion of activation was determined using the AgilentBioanalyser 2100, a chip based apparatus for conducting analyticelectrophoresis, using an Agilent Protein 80 kit (Agilent 5067-1515).The Analysis was performed using the manufacturers instructions providedin “Agilent 2100 Bioanalyzer 2100 Expert User's Guide”, Manual Partnumber: G2946-90000, Edition: November 2003 and “Agilent Protein 80 KitQuick Start Guide”, Part Number: G2938-90063, Edition 04/2007 (bothfrom: Agilent Technologies, Deutschland GmbH, Hewlett-Packard-Straβe 8,76337 Waldbronn, Germany).

In a 500 microlitre eppendorf tube, to 4 microlitres of sample was added2 microlitres sample buffer from the protein 80 kit. The sample wasboiled for five minutes in a heating block at 100° C. The sample wasallowed to cool for 10 seconds before 15 seconds of centrifugation in apicofuge. 84 microlitres of purified water was added and the vial wasmixed. The method analysis was run according to the above mentionedmanufacturers instructions which are able to resolve: Heavy chainFVII(a), Light Chain FVII(a), and single chain FVII which elute in thatrespective order. The proportion of activation is the ratio betweenHeavy chain FVII(a) (HC)+Light Chain FVII(a) (LC) relative to the totalFVII (HC+LC+SC). For example:

Proportion of activation=(HC+LC)/(HC+LC+SC)*100%

EXAMPLES Example 1 Calculation of Reaction Time for the Activation ofHuman Factor VII

6.316 kg of V158D/E296V/M298Q-FVII(a) (Example 6 in WO 02/22776)solution containing 10 mM histidine, 12 mM CaCl₂, 60 mM NaCl, pH 6.0 (at5° C.) was measured by UV280 and had an absorbance of 2.31 AU, using a 1cm lightpath. The concentration was calculated using the molarabsorbance coefficient (0.7 g/kg*AU) to be 1.62 g/kg. The initialproportion of activation was determined to be 20% in accordance with theabove mentioned protocol.

The variables for the reaction were decided to be as follows:

required proportion of activation: 95%;

pH: 6.50;

temperature: 21.5° C.

The activation time was calculated in accordance with the equation offormula (I) to be 128 minutes.

The activation reaction was started by adjusting pH upwards to 6.50 (at21.5° C.) using 25 ml of 1M NaOH. After 128 mins, the pH was loweredagain using 22 ml 1M HCl to 5.80 (22.3° C.). After ending the activationreaction, a sample was subjected to analysis, using the above mentionedmethodology, which reported the proportion of activation to be 95.6%.

The actual proportion of activation varied from that predicted by theequation of formula (I) by only 0.6% following over 2 hours of enzymaticactivation.

Example 2 Further Calculations of Reaction Time for the Activation ofHuman Factor VII

This experiment was performed on 5 separate purified batches ofV158D/E296V/M298Q-FVII(a) (Example 6 in WO 02/22776) each havingdifferent values of initial proportion of activation to assess theconsistency and accuracy of the method of the invention. This experimentwas performed in an analogous manner to that described in Example 1 andthe results can be seen in Table 1.

TABLE 1 Variable Batch 1 Batch 2 Batch 3 Batch 4 Batch 5 Initial 61 7453 16 34 Proportion of Activation Concentration 1.96 1.93 1.87 1.89 2.1(g/L) Required 92 96 94 96 98 Proportion of Activation pH 6.50 6.52 6.516.50 6.51 Activation 51 52 71 126 108 Time (min) Actual final 90 96 9696 98 Proportion of activation

The results from this assessment demonstrate that in 3 out of the 5batches, the method of the invention predicted the final proportion ofactivation exactly. In the remaining 2 batches, the variation was only2% which is not considered a significant or detrimental variation. Thus,table 1 shows cross validation of the model of the current invention.Experimental data is compared to the data that the model predicted in 5different production batches in a pilot project. With highly variableinput (16-74% active protease, “act time zero”), a constant output canbe predicted and obtained.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law),regardless of any separately provided incorporation of particulardocuments made elsewhere herein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. For example, the phrase the compound”is to be understood as referring to various “compounds” of the inventionor particular described aspect, unless otherwise indicated.

Unless otherwise indicated, all exact values provided herein arerepresentative of corresponding approximate values (e.g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate).

The description herein of any aspect or aspect of the invention usingterms such as “comprising”, “having,” “including,” or “containing” withreference to an element or elements is intended to provide support for asimilar aspect or aspect of the invention that “consists of”, “consistsessentially of”, or “substantially comprises” that particular element orelements, unless otherwise stated or clearly contradicted by context(e.g., a composition described herein as comprising a particular elementshould be understood as also describing a composition consisting of thatelement, unless otherwise stated or clearly contradicted by context).

1. A method of activating a serine protease blood coagulation factorwhich comprises the steps of: (a) measuring the initial concentration ofthe serine protease blood coagulation factor; (b) measuring the initialproportion of activated serine protease blood coagulation factor; (c)calculating the serine protease blood coagulation factor activationreaction time by correlation of the values measured in each of steps (a)and (b) with a value of required proportion of activated serine proteaseblood coagulation factor; and (d) performing the serine protease bloodcoagulation factor activation reaction for the time calculated in step(c); (e) terminating the reaction after the reaction time calculated instep (c).
 2. The method according to claim 1, wherein the reaction time,T, in step (c) is calculated in accordance with formula (I):$\begin{matrix}{t = \frac{- {\ln \left( \frac{{akt}\; {0 \cdot \left( {{akt} - 1} \right)}}{{akt} \cdot \left( {{{akt}\; 0} - 1} \right)} \right)}}{{{k(T)} \cdot {xb} \cdot F}\; 0}} & (I)\end{matrix}$ wherein “akt” refers to the required proportion of cleavedpolypeptide, “akt0” refers to the initial proportion of cleavedpolypeptide measured in step (b), “F0” refers to the initialconcentration of the polypeptide (in g/l) measured in step (a), k(T)refers to the reaction constant for the given reaction (in L/g/min) as afunction of temperature, T, and xb refers to the molar fraction.
 3. Themethod according to claim 2, wherein k(T) is a polynomial or a spline.4. The method according to claim 1 wherein the correlation proceduredescribed in step (c) is calculated in accordance with formula (II):$\begin{matrix}{t = \frac{- {\ln \left( \frac{{akt}\; {0 \cdot \left( {{akt} - 1} \right)}}{{akt} \cdot \left( {{{akt}\; 0} - 1} \right)} \right)}}{{k \cdot {xb} \cdot F}\; 0}} & ({II})\end{matrix}$ wherein “akt” refers to the required proportion of cleavedpolypeptide, “akt0” refers to the initial proportion of cleavedpolypeptide measured in step (b), k is the reaction constant, xb refersto the molar fraction, “F0” refers to the initial concentration of thepolypeptide (in g/l) measured in step (a).
 5. The method according toclaim 4, wherein k=0.29.
 6. The method according to claim 2 or claim 4,wherein xb=1.
 7. A method of preventing degradation of an activatedserine protease blood coagulation factor, said method comprising thesteps of: (a) measuring the initial concentration of the serine proteaseblood coagulation factor; (b) measuring the initial proportion ofactivated serine protease blood coagulation factor; (c) calculating theserine protease blood coagulation factor activation reaction time bycorrelation of the values measured in each of steps (a) and (b) with avalue of required proportion of activated serine protease bloodcoagulation factor; and (d) performing the serine protease bloodcoagulation factor activation reaction for the time calculated in step(c); (e) terminating the reaction after the reaction time calculated instep (c).
 8. The method according to claim 1, wherein said serineprotease is factor VII, a factor VII analogue or derivative thereof, orfactor IX.
 9. The method according to claim 8 wherein said factor VIIanalogue is V158D/E296V/M298Q-FVII(a).
 10. The method according to claim1 wherein the required proportion of cleavage will be between 90 and99%, between 94 and 99%, between 95 and 97%, between 96 and 98%, orbetween 97 and 99%.
 11. The method according to claim 1 wherein thecleavage reaction additionally comprises the addition of calcium ions.12. A method as defined in claim 1 wherein the activation reaction isperformed at a pH of between 6.0 and 8.0.
 13. A method as defined inclaim 1 wherein termination comprises lowering the pH to a value belowabout 6.0.
 14. A serine protease obtainable according to the method asdefined in claim
 1. 15. A composition comprising an activated factorVII(a) analogue, or derivative thereof, obtainable according to themethod as defined in claim 1.